The Protective Effects of LongTerm Oral Administration of

The Protective Effects of Long-Term Oral
Administration of Marine Collagen Hydrolysate
from Chum Salmon on Collagen Matrix
Homeostasis in the Chronological Aged
Skin of Sprague-Dawley Male Rats
Jiang Liang, Xinrong Pei, Zhaofeng Zhang, Nan Wang, Junbo Wang, and Yong Li
Abstract: To investigate the long-term effects of marine collagen hydrolysate (MCH) from Chum Salmon skin on the
aberrant collagen matrix homeostasis in chronological aged skin, Sprague-Dawley male rats of 4-wk-old were orally
administrated with MCH at the diet concentrations of 2.25% and 4.5% for 24 mo. Histological and biochemical analysis
revealed that MCH had the potential to inhibit the collagen loss and collagen fragmentation in chronological aged skin.
Based on immunohistochemistry and western blot analysis, collagen type I and III protein expression levels in MCHtreated groups significantly increased as compared with the aged control group. Furthermore, quantitative real-time
polymerase chain reaction and western blot analysis showed MCH was able to increase the expressions of procollagen
type I and III mRNA (COL1A2 and COL3A1) through activating Smad signaling pathway with up-regulated TGF-βRII
(TβRII) expression level. Meanwhile, MCH was shown to inhibit the age-related increased collagen degradation through
attenuating MMP-1 expression and increasing tissue inhibitor of metalloproteinases-1 expression in a dose-dependent
manner. Moreover, MCH could alleviate the oxidative stress in chronological aged skin, which was revealed from the
data of superoxide dismutase activity and the thiobarbituric acid reactive substances level in skin homogenates. Therefore,
MCH was demonstrated to have the protective effects on chronological skin aging due to the influence on collagen
matrix homeostasis. And the antioxidative property of MCH might play an important role in the process.
Keywords: chronological aging, collagen hydrolysate, collagen matrix, skin, Sprague-Dawley rat
Introduction
H: Health, Nutrition &
Food
Compared with the ultraviolet-induced or extrinsic skin aging, chronological or intrinsic skin aging is a cumulative process
depending on the passage of time (Fisher and others 2002). Histologically, chronological aged skin is characterized by the decreased
thickness of dermal matrix (El-Domyati and others 2002). The
principal structural components of extracellular matrix in dermis
consist of fibril-forming collagens. The predominant form of collagen in dermis is type I (85% to approximately 90%), followed
by small amounts of type III (10% to approximately 15%) (Chung
and others 2001). Type I collagen is characterized by thick fiber
that confer stiffness and resistance to perform a crucial function
in maintaining the structure of dermis. Whereas collagen type III
is characterized by thin fiber that present the resiliency of skin.
Collagen fibers arrange parallel to skin surface and are responsible
for the high tensile strength and resiliency of skin (Tayebjee and
others 2003).
MS 20100003 Submitted 1/2/2010, Accepted 5/5/2010. Authors Liang, Zhang,
Wang, Wang, and Li are with Dept. of Nutrition and Food Hygiene, School of Public
Health, Peking Univ., Beijing 100191, P.R. China. Author Pei is with Center for
Food and Drug Safety Evaluation, Capital Medical Univ., Beijing 100069, China.
Direct inquiries to author Li (E-mail: [email protected]).
H230
Journal of Food Science r Vol. 75, Nr. 8, 2010
In sun-protected young skin, the synthesis and degradation of
collagen are balanced to maintain the collagen content and structural integrity of skin. The collagen biosynthesis can be primarily
regulated by a family of transforming growth factor-β (TGFβ) through the Smad signaling pathway. The Smads are a series
of proteins that perform downstream functions from the serine/threonine kinase receptors of the TGF-β family, thereby propagating signals to the nucleus. The combination of TGF-β to the
TGF-β receptor type II (TβRII) is the initial step to activate the
intrinsic serine/threonine kinase activity of the TGF-β receptor
type I that triggers the phosphorylation and activation of Smad2
and Smad3. Meanwhile, the activation of Smad2 and Smad3 are
antagonized by the endogenous negative regulators Smad6 and
Smad7 (Moustakas and others 2001). The degradation of type I
and III fibrillar collagens is initiated by matrix metalloproteinases-1
(MMP-1), which belongs to the matrix metalloproteinases
(MMPs), a large family of zinc-dependent endo-proteases with a
broad range of substrate specificities and the capacity of degrading
all extracellular matrix proteins. On the other hand, the activity
of MMP-1 is inhibited by the specific endogenous tissue inhibitor
of metalloproteinases-1 (TIMP-1) (Visse and Nagase 2003). In
the chronological aged skin, collagen homeostasis is aberrant with
molecular features of the decreased synthesis of type I and III procollagens (the precursors to collagens) and elevated degradation
of mature collagen fibers, which result in general atrophy of the
R
C 2010 Institute of Food Technologists
doi: 10.1111/j.1750-3841.2010.01782.x
Further reproduction without permission is prohibited
Skin protective effect of collagen . . .
tive use of collagen hydrolysate from Chum Salmon (O. keta) skin
and explore its potentials in commercial applications, as antiskinaging functional ingredients in foods, cosmetics, and biomedical
materials.
Materials and Methods
Preparation and identification procedure of test substance
MCH provided by CF Haishi Biotechnology Ltd. Co. (Beijing, China) was prepared from the skin of wild-caught Chum
Salmon (O. keta) (from the East China Sea, average body weight,
1.47 kg). The procedures for MCH preparation and identification
were according to the method described in our previous research
(Pei and others 2010). Briefly, the fish skin was cleaned, scaled,
cut into small pieces, defatted, and homogenized. After being
homogenized and emulsified in distilled water, the material was
hydrolyzed by complex protease (3000 U/g protein) including 7%
of trypsin, 65% of papain, and 28% of alkaline proteinase, at 40 ◦ C
and pH 8 for 3 h. The resultant hydrolysate was reextracted by
centrifugation and subsequently separated through ceramic membrane (200 μm). After being purified through a procedure of
nanofiltration and condensed by cryoconcentration under vacuum at 70 ◦ C with an evaporation rate of 500 kg/h, decolorized
with active carbon at 75 ◦ C for 1 h, filtrated and dried by spray
drying with the pressure of 20 MPa at an evaporation rate of 200
kg/h, MCH powder used in the following investigations was obtained. Then the molecular weight distribution of the sample was
analyzed by the coupling technique of high-performance liquid
chromatography (HPLC, Waters Corp., Milford, Mass., U.S.A.)
and LDI-1700 matrix-assisted laser desorption ionization timeof-flight mass spectrometry (MALDI-TOF-MS) (Liner Scientific
Inc., Reno, Nev., U.S.A.).The amino acid composition was analyzed with an amino acid analyzer (H835-50; Hitachi, Tokyo,
Japan).
Animals
Young weaning male S-D rats, 4-wk-old, weighing 80 to approximately 90 g, were acquired from the Animal Service of
Health Science Center, Peking Univ.. Two rats per plastic cage
were housed with free access to chow and tap water and maintained in a filter-protected air-conditioned room with controlled
temperature (25 ± 2 ◦ C), relative air humidity (60 ± 5%) and 12
h light/dark cycles (light on 07:30–19:30 h). After the acclimatization period of 1 wk, the animals were randomly distributed
into 3 groups, that is, the vehicle control group (n = 20), fed
with the AIN93M rodent chow from Vital River Ltd., Co. (Beijing, China) comprising 25% of crude protein, 4.24% of crude
fat, and 55.19% of carbohydrate. The animals of 2 experimental
groups (n = 20 per group) were treated with MCH added in
the AIN93M diet, in which the equivalent proportion of crude
protein was replaced by MCH at the concentration of 2.25% and
4.5% (wt/wt), respectively. Body weight and food consumption
were recorded weekly in the first 6 mo and every 2 wk thereafter
until euthanasia. No mortality was found in any groups treated
with MCH for 12 mo. At the age of 25 mo, the survival rates of
control, 2.25% and 4.5% MCH groups were 62%, 70%, and 72%,
respectively. Ten rats from each group were anesthetized with an
intraperitoneal injection of pentobarbital sodium (45 mg/kg body
weight) and sacrificed. Moreover, during the experiment, another
group of male S-D rats (n = 10) fed with the control diet was
sacrificed at the age of 12 mo to be used as the middle-aged
controls.
Vol. 75, Nr. 8, 2010 r Journal of Food Science H231
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extracellular matrix and impaired collagen fibril organization (Rittie and Fisher 2002).
In addition, sustained age-associated oxidative damages to DNA,
proteins, and membrane lipids lead to the accumulation of thiobarbituric acid reactive substances (TBARs), the products of lipid peroxidation and other breakdown products from oxidatively modified proteins, carbohydrates, and nucleic acids (Lovell and others
1995; Tahara and others 2001). Meanwhile, the antioxidant defense is also attenuated in the chronological aged skin (Rittie and
Fisher 2002). Superoxide dismutase (SOD), which can effectively
scavenge reactive oxygen species (ROS) and inhibit lipid peroxidation, plays an important role in the maintenance of cellular
redox homeostasis and protects skin cells from oxidative damage
(Shindo and others 1993; Murakami and others 2009). Increased
oxidative stress can lead to the dysregulated intracellular and extracellular homeostasis that can modify cellular behavior and cell
matrix interactions. Moreover, ROS can directly induce MMPs
transcription and activate these enzymes (Nelson and Melendez
2004; Fisher and others 2009). Therefore, the age-related increased
oxidative stress is confirmed to play an important role in regulating
collagen matrix metabolism during skin aging (Yasui and Sakurai
2000; Siwik and others 2001).
As we know, collagen or gelatin (the partially hydrolyzed collagen) in dietary supplementation has been verified to involve in
the extracellular matrix synthesis for improving joint, nail, and
hair conditions (Moskowitz 2000; Zague 2008). As the further
polypeptide composite enzymatically hydrolyzed from gelatin,
collagen hydrolysate is characterized by relative lower molecular weight distribution (<6 kDa), higher absorption and bioavailability, as well as a wider range of functional and biological
properties, including antioxidant and antihypertensive activities
(Kim and others 2001; Morimura and others 2002). The collagen
hydrolysate can be traditionally isolated from the skin of landbased animals, such as cow and pig (Wang and others 2008). With
marine species comprising nearly one-half of the total global biodiversity, the skin, scale, and bone of the marine life, especially
the various fish species have become new sources of collagen hydrolysate (Wang and others 2008). In recent years, there is a growing interest in the potentially beneficial effects of marine collagen
hydrolysate (MCH) on skin aging. Although series of studies have
illustrated the antioxidative property of collagen hydrolysate from
marine fish contributed to the protective effects on UV-induced
skin photo-aging in vivo (Hou and others 2009; Zhuang and others
2009), the effects of marine fish collagen hydrolysate on intrinsic or chronological skin aging as well as the related molecular
mechanisms remain to be elucidated. Moreover, as we know, fish
collagen hydrolysates from different species, habitats and tissues
are significantly different in terms of properties because of the
unique amino acid composition and the variation in the amount
of amino acids (Wang and others 2008). At the present time, there
has been no report on the antichronological skin aging effects of
MCH from Chum Salmon, the deep sea fish widely distributed
in the north Pacific and Atlantic. Our previous study has revealed
the protective effect of 90-d oral ingestion of MCH from Chum
Salmon on D-galactose induced rapid intrinsic skin aging in mice
(Pei and others 2008). Further research still needs to be conducted
to confirm the long-term effect of MCH on chronological skin
aging. Therefore, in this study, we initially investigated the effects
of long-term (24 mo) oral ingestion of MCH from Chum Salmon
(Oncorhynchus keta) on the collagen content and collagen homeostasis in the chronological aged skin of male Sprague-Dawley
(S-D) rat. These experiments could help us to make more effec-
Skin protective effect of collagen . . .
After sacrifice, full thickness skin samples about 4 cm2 were immediately excised from the left depilated dorsal regions, trimmed
off loose connective tissue and musculature, snap-frozen in liquid
nitrogen, and then stored at −80 ◦ C until analysis. Full thickness
skin samples about 4 cm2 excised from the right depilated dorsal regions were fixed in 10% buffered formalin for histological
examinations and measurements.
All animals were handled in accordance with the guidelines
of the Principle of Laboratory Animal Care (NIH Publication
No. 85–23, revised 1985) and the guidelines of the Peking Univ.
Animal Research Committee.
Histological measurements
Skin samples fixed in 10% buffered formalin for at least 24 h
were followed by processing for conventional paraffin embedding.
Then the de-paraffinized sections of 5 μm thickness were processed for Masson’s trichrome stain to localize collagen. Dermal
thickness was determined by measuring the distance between the
epidermal–dermal junction and the dermal-fat junction at 5 randomly selected fields (40× microscopic magnifications) from 3
slides per sample in each animal using Image pro-plus 6.0 (Media
Cybernetics, Inc., Bethesda, Md., U.S.A.). To prevent observer
bias, all specimens were coded and examined without knowledge
of experimental conditions.
Assessment of total collagen by hydroxyproline method
The total collagen content was calculated using the assumption
that hydroxyproline accounts for 13% of collagen (Babraj and others 2005). Skin samples stored at −80 ◦ C were defatted and dried
to a constant weight. Determination of hydroxyproline content in
the dried skin sample from dorsal region was carried out according to the protocol of a commercial hydroxyproline detection kit
(Nanjing Jiancheng Biotechnology Co., Nanjing, China). Briefly,
the samples were hydrolyzed by sodium hydroxide for 20 min
at 100 ◦ C and oxidized by chloramine-T. The oxidized products
could react with dimethyl-amino-benzaldehyde to form the endproduct with the maximal absorption at 550 nm. The amount of
hydroxyproline in each sample was calculated by comparison with
the absorbance of the hydroxyproline standard and was expressed
as microgram hydroxyproline per milligram dry tissue sample.
H: Health, Nutrition &
Food
Immunohistochemistry
Immunohistochemistry was performed on 10% buffered formalin solution fixed, paraffin embedded tissue sections using a
Real Envision Detection ABC kit (Code nr Gk500705; GeneTech
[Shanghai] Company Ltd, Shanghai, China). The detailed procedure was performed according to the manufacturers’ instructions.
In brief, the deparaffinized and transferred 5 μm thickness slides
were incubated in a mixture of methanol and 3% hydrogen peroxide to block the activity of endogenous peroxidase. For antigen
retrieval, the slides were boiled in 0.01 mmol/L sodium citrate
(pH 6.0) buffer solutions for 15 min. To reduce nonspecific background staining, slides were incubated in goat serum albumin at
room temperature for 30 min. Then the sections were incubated
with the primary antibodies (mouse monoclonal anti-Collagen I
at 1:700 [Abcam, Cambridge, UK] and mouse monoclonal antiCollagen III at 1:500 [Abcam]) at 4 ◦ C overnight. The sections
were then incubated at room temperature with goat-anti-mouse
EnVision–HRP (Horseradish Peroxidase)–enzyme for 120 min.
The immunoreactivity was visualized with 3,3 -diaminobenzidine
chromogens. The sections were counterstained with hematoxylin
H232 Journal of Food Science r Vol. 75, Nr. 8, 2010
and viewed under an Olympus BH-2 microscope (Olympus
Corp., Tokyo, Japan) with images obtained by digital capture.
Real-time quantitative PCR analysis
The isolation of total RNA from the skin sample (about 50 mg)
was according to the manufacturers’ protocol of the RNeasyR
fibrous tissue mini kit (Qiagen). Total RNA was quantified by
30 to 50 μL RNase-free ddH2 O and analyzed for quantity and
quality using a spectrophotometer.
Total RNA was reverse transcribed in a 25 μL reaction using the M-MLV Reverse Transcriptase (Promega
M170A). Two microliters of the resulting cDNA was then
used in the real-time PCR step that was performed using
BioEasy SYBR Green I Real Time PCR Kit Manual in
the Line-gene fluorescence quantitative PCR detection system
(Hangzhou Bioer Technology Co., Ltd., Hangzhou, China).
The primers used were as follows: Rattus β-actin (NM031144.2, 150 bp), 5 -CCCATCTATGAGGGTTACGC-3 and
5 -TTTAATGTC ACGCACGATTTC-3 ; Rattus COL1A2
(NM-053356, 118 bp), 5 -GCAGTGTGCAATATGATCCA-3
and 5 -TTGACAATGTCCACAACAGG-3 ; Rattus COL3A1
(X70369, 81 bp), 5 -CCATTGCTGGAGTTGGAGGT-3
and 5 -TGTTGATCTTGAAATCCATTGGAT-3 . The relative
change in mRNA expression level was determined by the 2Ct
method with β-actin as the reference gene.
Western blot analysis
Frozen skin sample (about 100 mg) was cut into small pieces
(about 1 mm2 ) and powdered in a liquid nitrogen bath. Added
with about 1 mL tissue lysis solution (Tris [50 mM], Triton X-100
[0.1%], NaCl [150 mM], and EGTA [ethylene glycol tetraacetic
acid]/EDTA [ethylenediaminetetraacetic acid; 2 mM], pH 7.4,
containing mammalian protease inhibitor cocktail (1:100 dilution), sodium pyrophosphate [1 mM], phenylmethylsulfonyl fluoride [PMSF; 10 μg/mL], sodium vanadate [1 mM], and sodium
fluoride [50 mM]), the sample was homogenized with tissue homogenizer (XHF-1, Ningbo Scientz Biotechnology Co., Ltd.,
Ningbo, China) for three 5 s pulses at 5000 rpm in an ice bath.
Homogenates were then centrifuged at 12500 rpm for 5 min
at 4 ◦ C to collect the supernatants. Total protein concentration
was determined by the BCA assay kit (Pierce Biotechnology, Ill.,
IL USA.). Equal amounts (40 μg) of total protein per sample
and prestained molecular weight standards were separated by Trisglycine gels (Collagen I, 8% gel; Collagen III, 10% gel; TGFβR
II, 10% gel; Smad2, 10% gel; phospho-Smad2, 10% gel; Smad3,
10% gel; phospho-Smad3, 10% gel; Smad7, 12% gel; TIMP-1,
15% gel; MMP-1, 10% gel; β-actin, 10% gel) and transferred
to polyvinylidene fluoride (PVDF) membranes (Millipore, Mass.,
USA.).
The membranes were blocked by 5% skim milk powder in Trisbuffered saline with 0.1% Tween-20 for 2 h at room temperature
and incubated overnight at 4 ◦ C with the following antibodies: mouse monoclonal anti-Collagen I (1:1500; Abcam), mouse
monoclonal anti-Collagen III (1:500; Abcam), rabbit polyclonal
anti-TGFβRII (1:500; Santa Cruz Biotechnology, Inc., Santa
Cruz, Calif., U.S.A.), goat polyclonal anti-Smad2 (1:200; Santa
Cruz), rabbit polyclonal antiphospho-Smad2 (1:200; Santa Cruz),
mouse monoclonal anti-Smad3 (1:200; Santa Cruz), rabbit polyclonal antiphospho-Smad3 (1:1000; Millipore); goat polyclonal
anti-Smad7 (1:200; Santa Cruz), rabbit polyclonal anti-MMP-1
(1:100; Boster, Wuhan, China), rabbit polyclonal anti-TIMP-1
(1:200; Santa Cruz, CA, USA), and rabbit polyclonal anti-β-actin
Skin protective effect of collagen . . .
MCH intake and bodyweight
Treated with MCH for 24 mo, daily food consumption did
not significantly differ among the aged control (43.7 ± 3.9
g/kg.bw/d), 2.25% MCH-treated (44.8 ± 3.2 g/kg.bw/d) and
4.5% MCH-treated (44.4 ± 3.8 g/kg.bw/d) aged groups. During
the study, the intake of MCH in aged control and aged MCHtreated groups were estimated to be 0, 1.008, 1.998 g/kg.bw/d,
respectively. Bodyweights of rats submitted to MCH treatment
were similar to those of the control rats at the beginning (controls:
93.00 ± 13.46 g; 2.25% MCH: 93.30 ± 11.45 g; 4.5% MCH:
94.00 ± 14.79g; P > 0.05) and at the end of the study (controls: 886.67 ± 137.68 g; 2.25% MCH: 843.88 ± 109.42 g; 4.5%
Measurement of oxidative status in skin
About 100 mg skin sample was homogenized with 9 volumes MCH: 934.00 ± 65.05 g; P > 0.05), indicating that long-term
(w/v) of phosphate buffered saline (PBS, pH 7.0) using tissue administration of MCH did not notably affect their appetite or
homogenizer (XHF-1, Ningbo Scientz Biotechnology Co., Ltd.) provoke malnutrition.
for three 5 s pulses at 5000 rpm at 4 ◦ C. The SOD activity
and TBARSs level in the supernatant of tissue homogenates were Long-term effects of MCH on collagen accumulation and
determined according to the manufacturers’ instructions of the collagen fiber morphology in the chronological aged skin
The dermis thickness was determined based on Masson’s
commercial kits purchased from Jiancheng Inst. of Biotechnology
trichrome stained dorsal skin section with 40 times of magnifi(Nanjing, China).
cation. As shown in Figure 1A and 1B, the dermis thickness of
middle-aged control rats was about twice that of aged control
Statistical analysis
rats (middle-aged controls: 6501.10 ± 582.98 μm; aged controls:
Statistical analyses were performed using SPSS (Version 13.0,
3340.52 ± 448.35 μm; P < 0.001). Compared with the aged
SPSS Inc., Ill., U.S.A.). All variances in the measurement data
control group, long-term application of MCH dose-dependently
expressed as mean ± standard error of mean (SEM) were checked
increased the dermis thickness with significance in 4.5% MCH
for homogeneity by the Bartlett’s test. The one-way ANOVA
group (4.5% MCH: 4108.23 ± 314.48 μm, P < 0.05). As a
test was used to analyze the group difference by least significant
quantitative measure of total collagen, the hydroxyproline content
difference (LSD) (equal variance assumed) or Tamhane’s T2 (equal
was determined. The result in Figure 1C indicated a significant
variances not assumed) post hoc tests. A value of P < 0.05 was
age-related decrease of hydroxyproline content in skin (middleconsidered significant.
aged controls: 17.98 ± 3.08 μg/mg, aged controls: 12.02 ± 2.20
μg/mg, P < 0.001). The hydroxyproline content in the 4.5%
Results
MCH-treated group was 1.26-fold higher than that in the vehicletreated aged group (4.5% MCH: 15.14 ± 2.09 μg/mg, P < 0.05).
Characterization of MCH
The analysis of MCH indicated 86.5% of peptides with the As a whole, the increased dermal thickness and total collagen
molecular weights distributed between 130 and 1000 Da, and content in MCH-treated chronological aged skin indicated the
91.5% below 1000 Da. The result of amino acid composition positive effect of MCH on collagen accumulation.
Meanwhile, we examined the effect of MCH on the morpholin Table 1 shows that MCH was rich in Gly > Glu > Pro >
ogy
alteration of collagen fibers in the chronological aged skin. As
Hyp > Asp > Ala > Arg. Furthermore, MCH was revealed to
contain very little or no carbohydrate by negative staining the shown in the Masson’s trichrome stained sections with 200 times
polyacrylamide gel with periodic acid-Schiff reagent (data not of magnification (Figure 1A), collagen fibers in the dermis of aged
vehicle control group showed weak blue staining and appeared to
shown).
be more sparsely, fragmented, and disorganized versus the middleaged control group. Compared with the aged control group, collaTable 1– Amino acid composition of MCH from Chum Salmon gen fibers of the MCH-treated groups were obviously denser and
skin.
more systematic with a dose-dependent tendency. Thus MCH was
Amino acid
Nr residues/100 residues revealed with the inhibitory effect on collagen fragmentation in
the chronological aged skin.
Glycine
Glutamic acid
Proline
Hydroxyproline
Aspartic acid
Alanine
Arginine
Lysine
Leucine
Serine
Valine
Isoleucine
Threonine
Phenylalanine
Histidine
Methionine
Tyrosine
23.77
12.22
9.79
7.51
7.29
6.59
6.08
5.66
4.64
4.23
2.94
2.57
2.53
2.51
1.61
0.03
0.03
Long-term effects of MCH on the expressions of collagen
type I and collagen type III in the chronological aged skin
Further investigations on the effects of MCH on the levels of
collagen type I and collagen type III were performed by immunohistochemistry, western blot, and quantitative real-time PCR analysis, respectively.
As shown in Figure 2A, skin sections are immune stained with
antibody for collagen type I and collagen type III in the area of
extracellular matrix of dermis. The type I collagen fibers with
dense distribution were stained strong brown whereas the type
III collagen fibers were showed as faint brown with sparse distribution. Compared with the middle-aged control group, the
2 types of collagen fibers in the vehicle-aged control group
Vol. 75, Nr. 8, 2010 r Journal of Food Science H233
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(1:200; Santa Cruz, CA, USA). After being washed in Tween-TBS
(the self-made reagent of Tris Buffered Saline with 0.05%TweenR
20), membranes were incubated for 1 h at 37 ◦ C with the appropriate HRP-conjugated secondary antibodies. The target proteins
were visualized using enhanced chemiluminescence reagents (ECL
kit; Millipore) and developed using Kodak films. The intensity of
each band was quantified using public domain National Institutes
of Health (NIH) Image Program (http://rsb.info.nih.gov/nihimage/download.html) and normalized using β-actin as a marker
for equal protein loading.
Skin protective effect of collagen . . .
showed weaker staining and looser distribution. However, longterm MCH-treated chronological aged skin showed greater staining and denser distribution of collagen fibers.
Consistent with the immunohistochemical profile, the western
blot analysis showed in Figure 2B revealed that the collagen type I
and type III expression levels in the vehicle-treated aged controls
significantly decreased to 37.9 ± 7.5% and 42.2 ± 8.5% of the
middle-aged controls (P < 0.001), respectively. Compared with
the vehicle-aged control group, the expression levels of collagen
type I in 2.25% and 4.5% MCH-treated groups were 1.84 ±
0.36 and 1.96 ± 0.31 times of the aged control group (P < 0.01),
respectively. Similarly, MCH administration significantly increased
the expression of collagen type III in 2.25% and 4.5% MCHtreated groups to 1.38 ± 0.27 times (P < 0.05) and 1.92 ± 0.22
times of the aged controls (P < 0.001), respectively. Therefore,
long-term treatment of MCH was indicated to markedly increase
the expression levels of collagen type I and collagen type III in the
chronological aged skin.
Next, the effects of MCH treatment on the mRNA levels of
procollagen type I (COL1A2) and III (COL3A1) were revealed
from the quantitative real-time PCR analysis (Figure 2C). Both
the mRNA levels of COL1A2 and COL3A1 in the chronological
aged skin decreased to 41.0 ± 10.1% and 47.8 ± 8.5% of the
middle-aged controls, respectively (P < 0.001). Administration
with MCH of 2.25% and 4.5% dose-dependently promoted the
synthesis of type I and III procollagen in chronological aged skin.
In 2.25% and 4.5% MCH-treated groups, the level of COL1A2
increased to 1.38 ± 0.2 times (P < 0.05) and 1.68 ± 0.29 times
(P < 0.001) of the aged controls, and the level of COL3A1 was
1.29 ± 0.23 times (P < 0.05) and 1.38 ± 0.17 times (P < 0.01)
of the aged controls, respectively.
From these results, we found that long-term oral administration
of MCH could promote the expressions of collagen type I and
type III proteins as well as increase the procollagen mRNA levels
in chronological skin.
H: Health, Nutrition &
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Figure 1–The effects of long-term oral administration of MCH on the dermal thickness, collagen content, and collagen morphology in the chronological
aged skin of S-D male rats. (A) Representative skin sections stained by Masson’s trichrome to assess the dermal thickness (original magnification
40×), the collagen content, and fiber morphology (original magnification 200×). Masson’s trichrome stains collagen fibers blue. Dermal thickness
was determined by measuring the distance between the epidermal–dermal junction and the dermal-fat junction at 5 randomly selected fields (40×
microscopic magnifications) from 3 slides per sample in each animal. The Masson’s trichrome stained sections with 200 times of magnification showed
the morphology and density of collagen fibers. (B) Comparisons of the dermal thickness of S-D male rats. Bars represent the mean ± SEM of the
dermal thickness from at least 6 rats of each group. Values were determined at 5 randomly selected sites/fields and analyzed by one-way analysis of
variance (ANOVA). ∗ P < 0.05 and ∗∗∗ P < 0.001 versus the aged control group. ### P < 0.001 versus the middle-aged control group. (C) Comparisons
of hydroxyproline content in skin of S-D male rats. Bars represent the mean ± SEM of the hydroxyproline content from 10 rats of each group. Values
are expressed as microgram hydroxyprolline per milligram sample and were analyzed by one-way ANOVA.∗ P < 0.05 and ∗∗∗ P < 0.001 versus the aged
control group. # P < 0.05, ## P < 0.01, and ### P < 0.001 versus the middle-aged control group. (By Jiang Liang, Xinrong Pei, Zhaofeng Zhang, Nan Wang,
Junbo Wang, and Yong Li.)
H234 Journal of Food Science r Vol. 75, Nr. 8, 2010
Skin protective effect of collagen . . .
skin were revealed from the comparisons between the middle-aged
control group and the aged control group (Smad2 of middle-aged
controls: 1.36 ± 0.15 times of aged controls, P < 0.01; Smad3
of middle-aged controls: 1.20 ± 0.07 times of aged controls, P <
0.05). Although no noticeable effect on the Smad2 and Smad3
protein levels was indicated from the comparison between MCHtreated groups and the aged control group (P > 0.05), MCH
showed a remarkable effect on the phosphorylation of Smad2 and
Smad3. As shown in Figure 3B, the ratio of p-Smad2/Smad2 in
2.25% and 4.5% MCH-treated groups significantly increased 1.28
and 1.16 times of the aged control group (P < 0.05), respectively.
Similarly, the ratio of p-Smad3/Smad3 in 2.25% and 4.5% MCHtreated groups significantly increased 1.23 and 1.18 times of the
aged control group (P < 0.05), respectively.
Smad7 acts as the inhibitory Smad (I-Smad) with the activity
to inhibit the signaling function of receptor-activated Smads thus
interfere with the TGF-β/Smad signaling pathway. Figure 3B
Figure 2–The effects of long-term oral administration of MCH on the levels of collagen type I and III proteins and procollagen mRNAs in the chronological
aged skin of S-D male rats. (A) Representative skin sections treated with anti-rat collagen type I mouse antibody and anti-rat collagen type III mouse
antibody showing the distribution of collagen type I and type III fibers in the dermis (original magnification 100×). Skin sections were immune stained
with antibody for collagen type I and collagen type III in the area of extracellular matrix of dermis. The type I collagen fibers with dense distribution
were stained strong brown whereas the type III collagen fibers were showed as faint brown staining with sparse distribution. (B) Representative western
blot results showing the protein levels of collagen type I and III in the skin of (1) middle-aged control group, (2) aged control group, (3) aged MCH
2.25%, and (4) aged MCH 4.5% groups. The relative density of the bands was normalized to β-actin. Bars representing values as fold changes of aged
control group are the mean ± SEM of at least 6 samples of each group. Values were analyzed by one-way ANOVA. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P <
0.001 versus the aged control group. # P < 0.05, ## P < 0.01, and ### P < 0.001 versus the middle-aged control group. (C) Real-time quantitative PCR
gene expression measurements of procollagen type I (COL1A2), procollagen type III (COL3A1). Gene expression values expressed as fold changes of
aged controls are the mean ± SEM of at least 6 samples of each group. Values were analyzed by one-way ANOVA. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P <
0.001 versus the aged control group. ### P < 0.001 versus the middle-aged control group. (By Jiang Liang, Xinrong Pei, Zhaofeng Zhang, Nan Wang,
Junbo Wang, and Yong Li.)
Vol. 75, Nr. 8, 2010 r Journal of Food Science H235
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Food
Long-term effects of MCH on the expressions of TGFβR II
and Smad proteins in the chronological aged skin
The binding of TGF-β to TβRII forming a complex is the
initial step of the TGF-β/Smad signaling cascade that regulates
procollagen synthesis and extracellular matrix production in skin
(Massague 1998). The results of Figure 3A revealed that the level
of TβRII significantly decreased in the aged vehicle control group
as compared with that of the middle-aged vehicle control group
(aged controls: 50.7 ± 5.8% of middle-aged controls, P < 0.001).
The level of TβRII considerably reduced in the MCH-treated
aged skin as compared with that of the aged controls (2.25% MCH:
1.42 ± 0.20 times of the aged controls, P < 0.01; 4.5% MCH:
1.35 ± 0.27 times of the aged controls, P < 0.001).
As the receptor-regulated transcription factors, Smad2 and
Smad3 mediate TGF-β signals when being phosphorylated
(Schiller and others 2004). In our study, age-related decreases in
the levels of Smad2 and Smad3 (Figure 3B) in chronological aged
Skin protective effect of collagen . . .
showed that the protein level of Smad7 in aged control group
significantly increased to 1.25 ± 0.10 times of middle-aged control
group (P < 0.01). Smad7 protein level in the 4.5% MCH-treated
chronological aged skin markedly decreased as compared with
that of the aged control group (4.5% MCH: 82.6 ± 9.3% of aged
controls, P < 0.05).
Long-term effects of MCH on the expressions of MMP-1
and TIMP-1 proteins in the chronological aged skin
Type I and III collagens are initially degraded by the matrix
metalloproteinases-1 (MMP-1), which activity can be inhibited
by the TIMP-1 (Nagase and Woessner Jr 1999). As shown in
Figure 4, the expression level of MMP-1 in the aged control
group increased 2-fold versus the middle-aged control group (P <
0.001). Long-term oral ingestion of 4.5% MCH in diet was shown
to significantly inhibit the increased MMP-1 expression level in
the chronological aged skin (4.5% MCH: 67.4 ± 9.4% of aged
controls, P < 0.001). As the specific inhibitors of MMP-1, the
level of TIMP-1 showed no significant age-related difference from
the comparison between the middle-aged control group and aged
control group (P > 0.05). While 4.5% MCH treatment significantly elevated the expression level of TIMP-1 in the chronological aged skin (1.37 ± 0.19 times of aged controls, P < 0.05).
Long-term effects of MCH on the oxidative stress in the
chronological aged skin
The oxidative stress in the chronological aged skin was assessed
by the activity of SOD and the level of lipid peroxidation product (TBARS) in skin homogenates. As the Table 2 indicated,
compared with the middle-aged control group, the SOD activity
in the skin homogenates of aged control group significantly decreased while the TBARS level significantly increased (P < 0.01).
The TBARS levels in the chronological skin homogenates of
MCH-treated groups dose-dependently decreased, with significance in 4.5% MCH group (P < 0.05). MCH treatment resulted
in an elevation in the activity of SOD enzyme, while no statistical
difference was revealed (P > 0.05).
Discussion
Marine proteins are likely to contain a lot of specific and potent
bioactive subsequences in nutritional perspective for their competitive and aggressive living conditions (Aneiros and Garateix 2004).
With various bioactive properties, MCH has gained increasing
popularity as ingredients of functional foods or pharmaceuticals
(Kim and Mendis 2006). To the best of our knowledge, the current study first revealed the effects of long-term oral administration
of collagen hydrolysate from Chum Salmon on the chronological
skin aging in vivo. Moreover, the effects of MCH on the primary
molecular mechanism of collagen synthesis and degradation were
elucidated.
In line with the characteristics of chronological aged skin (ElDomyati and others 2002; Varani and others 2006), the skin of
aged controls was suggested with significant decreased collagen
content and increased collagen fragmentation. In this study, longterm oral ingestion of MCH was found to have inhibitory effects
on the age-related decreases in dermal thickness, collagen content
as well as the expressions of collagen type I and III. Moreover,
compared with the aged controls, the denser and more systematic collagen fibers in the dermis treated with MCH indicated
the long-term oral administration of MCH might inhibit the increased degradation of collagen fibers in chronological aged skin.
As we know, both the quantity and quality of collagen fibers are
Table 2– The effects of long-term oral administration of MCH
on the SOD activity and TBARS level in chronological aged skin
homogenate of S-D male rats.
Group (MCH%)
n
SOD activity
(U/mg protein)
Middle-aged control
Aged control
Aged MCH 2.25%
Aged MCH 4.5%
10
10
10
10
128.83
96.13
102.77
104.50
±
±
±
±
12.05∗∗
24.93##
21.50#
15.58#
TBARS
(MDA nmol/
mg protein)
1.76
3.75
2.60
2.27
±
±
±
±
1.07∗∗
1.57##
1.17
1.11∗
Values represent the mean ± SEM, which were analyzed by one-way ANOVA test. ∗ P <
0.05 and ∗∗ P < 0.01 versus the aged control group. # P < 0.05 and ## P < 0.01 versus the
middle-aged control group. Ten rats were used in each group. (By Jiang Liang, Xinrong
Pei, Zhaofeng Zhang, Nan Wang, Junbo Wang, and Yong Li.)
H: Health, Nutrition &
Food
Figure 3–The effects of long-term oral administration of MCH on the levels of TβRII, Smad2, p-Smad2, Smad3, p-Smad3, and Smad7 proteins in the
chronological aged skin of S-D male rats. Representative western blot results showing the levels of TβRII (A), Smad2, p-Smad2, Smad3, p-Smad3, and
Smad7 (B) of (1) middle-aged control group, (2) aged control group, (3) aged MCH 2.25%, and (4) aged MCH 4.5% treated groups. The relative density
of the bands was normalized to β-actin. Bars representing values as fold changes of aged control group are the mean ± SEM of at least 6 samples of
each group. Values were analyzed by one-way ANOVA. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 versus the aged control group.# P < 0.05, ## P < 0.01,
and ### P < 0.001 versus the middle-aged control group. (By Jiang Liang, Xinrong Pei, Zhaofeng Zhang, Nan Wang, Junbo Wang, and Yong Li.)
H236 Journal of Food Science r Vol. 75, Nr. 8, 2010
Skin protective effect of collagen . . .
fect of MCH on the Smad signaling was revealed in this study.
It is well known that the phosphorylation of receptor-activated
Smad2 and Smad3 is an initial molecular event of Smad signaling
and can be prevented by I-Smads, as Smad6 and Smad7 (Rittie
and Fisher 2002; Quan and others 2004). In this study, the data
showed that the levels of phosphorylated Smad2 and Smad3 were
elevated in all MCH-treated groups, whereas the level of Smad7
was significantly attenuated in 4.5% MCH-treated groups. When
taken together, these data suggested that MCH had the potential
to promote procollagen synthesis in the chronological aged skin
with the mechanism involved in the activation of Smad signaling
induced by the increased TGF-β responsiveness.
Moreover, except reduction of procollagen biosynthesis, increased degradation of mature collagen fibers with a concomitant
increase of MMPs expression also contributes to the unbalanced
homeostasis in chronologically aged skin (Kim and others 2006).
Consistent with previous reports (Fisher and others 2002), the
present study showed the expression of MMP-1 protein in the skin
of aged control rats significantly increased as compared with the
middle-aged controls. Over-expression of MMP-1 can result in
the increased collagen fragmentation, which in turn promotes oxidative stress and elevates MMP-1 expression in fibroblasts (Varani
and others 2006; Fisher and others 2009). As the specific inhibitor
of MMP-1 activity, TIMP-1 expression in our study did not show
significant age difference, which was in accordance with the result of Fisher and others (2009). The long-term administration of
MCH at 4.5% concentration was suggested to significantly inhibit
the expression of MMP-1 and elevate the level of TIMP-1 in the
chronological aged skin. Therefore, the elevated MMP-1 protein
and reduced TIMP-1 protein levels together contributed to the
inhibitory effect of MCH on the degradation of collagen fibers in
chronological aged skin.
In addition, MCH treatment was shown to markedly inhibit
the lipid peroxidation product (TBARS) level in the aged skin
homogenates rather than increase the activity of SOD, which indicated MCH itself might have the antioxidant property. Lin and
Li (2006) found that the low-molecular-weight proteins and peptides, especially the fractions with molecular weights below 2000
Da, had high radical scavenging activity due to the relatively high
amounts of some amino acids such as Gly, Pro, and Ala. In our
study, the collagen hydrolysate rich in Gly > Glu > Pro > Hyp was
Figure 4–The effects of long-term oral
administration of MCH on the levels of MMP-1
and TIMP-1 proteins in the chronological aged
skin of S-D male rats. Representative western
blot results showing the levels of TIMP-1 (A)
and MMP-1 (B) of (1) middle-aged control
group, (2) aged control group, (3) aged MCH
2.25%, and (4) aged MCH 4.5% treated groups.
The relative density of the bands was
normalized to β-actin. Bars representing values
as fold changes of aged control group are the
mean ± SEM of at least 6 samples of each
group. Values were analyzed by one-way
ANOVA. ∗ P < 0.05 and ∗∗∗ P < 0.001 versus the
aged control group.# P < 0.05 and ### P < 0.001
versus the middle-aged control group. (By Jiang
Liang, Xinrong Pei, Zhaofeng Zhang, Nan Wang,
Junbo Wang, and Yong Li.)
Vol. 75, Nr. 8, 2010 r Journal of Food Science H237
H: Health, Nutrition &
Food
determined by the balance between collagen degradation and synthesis (Lee and others 2007). In this regard, long-term MCH
treatment was indicated to play an important role in the process
of collagen metabolism.
The level of collagen biosynthesis can be generally reflected
by the levels of type I and type III procollagen, the precursor molecules of mature collagen (Chung and others 2001). In
chronological aged skin, both the number of fibroblasts and their
capacity to synthesize procollagen were reduced as compared with
young skin (Varani and others 2000; Chung and others 2001).
In this study, the mRNA levels of procollagen type I (COL1A2)
and type III (COL3A1) were shown to decrease with age, which
indicate the potential of long-term oral administration of MCH
to promote the expressions of type I procollagen gene (COL1A2)
and type III procollagen gene (COL3A1). In line with the result,
collagen extract from scallop shell was reported to increase the
mRNA expression level of type I collagen in skin fibroblast cells
and activate the collagen metabolism (Torita and others 2007).
It is confirmed that the production of type I and type III procollagen and other components of the dermal extracellular matrix can be primarily regulated by TGF-β, which is a family
of profibrotic cytokines with the ability to exert potent stimulatory effects on collagen synthesis, extracellular matrix accumulation, and fibroblast proliferation through the cellular Smad
signal transduction pathway (Fisher and others 2002). TGFβ/Smad signaling pathway is verified to directly promote the
mRNA expressions of COL1A2 and COL3A1 (Chen and others
1999; Schiller and others 2004). The combination of TGF-β to
the TβRII is the initial step. TβRII then phosphorylates TβRI,
which can phosphorylate Smad proteins and propagate signals to
the nucleus (Massague 1998). As previous study described (Rittie
and Fisher 2002), TβRII protein level in this study was shown to
decrease with age. It is demonstrated that transcriptional downregulation of TβRII is primarily responsible for both the reduced
TGF-β responsiveness and the impairment of TGF-β/Smad pathway, which together contribute to the reduced procollagen synthesis in skin fibroblasts (Rittie and Fisher 2002; Quan and others
2004). Therefore, we speculate that the up-regulated expression
of TβRII in MCH-treated groups might resulted in the increased
TGF-β responsiveness and up-regulation of type I and type III
procollagen in the chronological aged skin. Furthermore, the ef-
Skin protective effect of collagen . . .
shown to be consisted of 91.5% of peptide fractions with molecular
weights below 1000 Da. Thus MCH in the present study is confirmed with relative high antioxidant activity. As previous study
described, the antioxidative ability of fish collagen hydrolysate is
verified to play an important role in anti-UV-B induced photoaging (Zhuang and others 2009). Hence, the antioxidative activity
of MCH was thought to be an important contributory factor affecting the aberrant extracellular homeostasis in the chronological
aged skin. Besides that, after oral ingestion, collagen hydrolysate
was verified to be degraded into peptide forms in plasma, such
as Pro-Hyp and Pro-Hyp-Gly peptides. These degraded oligopeptides were demonstrated in vitro studies to function as chemotactic stimuli for fibroblasts to trigger the synthesis of new collagen
fibers as well as the extracellular matrix reorganization in a proteinspecific manner (Iwai and others 2005; Zague 2008). However,
our data do not allow us to directly ascertain the chemotactic activity of MCH and further research is still needed to clarify the
functional peptide fragments in MCH.
Conclusions
The present study demonstrated that the protective effects of
long-term MCH on chronological skin aging may be due to affecting extracellular collagen homeostasis in the aged skin of S-D
male rats. MCH was verified to promote collagen type I and type
III synthesis through activating Smad signaling pathway with upregulated TβRII protein level. Meanwhile, MCH inhibited the
aged-related increased collagen degradation through attenuating
MMP-1 expression and elevating TIMP-1 expression in chronological aged skin. Moreover, the antioxidative property of MCH
might play an important role during the process. All evidences
earlier, taken together, lead us to propose that long-term oral administration of MCH from Chum Salmon might be a beneficial
method for slowing chronological skin aging.
Acknowledgments
The authors are grateful to the company CF Haishi Biotechnology Ltd. for providing the samples of MCH used in this study. This
research was supported by the foundation (nr 2006BAD27B08)
from the Ministry of Science and Technology of the People’s Republic of China.
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